U.S. patent application number 13/207197 was filed with the patent office on 2011-12-01 for detection device for detecting biological microparticles such as bacteria, viruses, spores, pollen or biological toxins, and detection method.
This patent application is currently assigned to EADS DEUTSCHLAND GMBH. Invention is credited to Alois FRIEDBERGER, Gerhard MULLER, Ulrich REIDT.
Application Number | 20110294113 13/207197 |
Document ID | / |
Family ID | 39809716 |
Filed Date | 2011-12-01 |
United States Patent
Application |
20110294113 |
Kind Code |
A1 |
FRIEDBERGER; Alois ; et
al. |
December 1, 2011 |
DETECTION DEVICE FOR DETECTING BIOLOGICAL MICROPARTICLES SUCH AS
BACTERIA, VIRUSES, SPORES, POLLEN OR BIOLOGICAL TOXINS, AND
DETECTION METHOD
Abstract
A device for the detection of micro particles that can be marked
by probes or antibodies capable of being detected by radiation has
a filter, a supply system, and a detection system. Fluid to be
examined is passed over a filter to filter out the micro particles
and to perform the marking steps by supplying corresponding marking
substances to the filter
Inventors: |
FRIEDBERGER; Alois;
(Oberpframmern, DE) ; REIDT; Ulrich;
(Schwalmstadt, DE) ; MULLER; Gerhard; (Grafing,
DE) |
Assignee: |
EADS DEUTSCHLAND GMBH
Ottobrunn
DE
|
Family ID: |
39809716 |
Appl. No.: |
13/207197 |
Filed: |
August 10, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12598483 |
Jan 28, 2010 |
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PCT/EP2008/055231 |
Apr 29, 2008 |
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13207197 |
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Current U.S.
Class: |
435/5 ; 435/6.11;
435/6.12; 435/7.32; 436/501 |
Current CPC
Class: |
G01N 33/56983 20130101;
C12M 1/3446 20130101; G01N 33/56911 20130101; C12Q 1/6888
20130101 |
Class at
Publication: |
435/5 ; 435/7.32;
435/6.11; 435/6.12; 436/501 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70; C12Q 1/68 20060101 C12Q001/68; G01N 33/53 20060101
G01N033/53; G01N 33/569 20060101 G01N033/569 |
Foreign Application Data
Date |
Code |
Application Number |
May 4, 2007 |
DE |
10 2007 021 387.7 |
Claims
1. A detection method to detect biological micro particles,
including living bacteria, viruses, and/or biological toxins,
comprising: passing a fluid containing the biological micro
particles through a filter configured to filter out the biological
micro particles; chemically marking the biological micro particles
adhering to the filter by specific probes configured to be detected
by radiation; and detecting the radiation correlating to the
probes.
2. The detection method according to claim 1, wherein a liquid
sample to be examined is pumped over the filter by a fluidic system
including at least one pump and at least one control valve when
passing the fluid containing the biological micro particles through
the filter.
3. The detection method according to claim 1, wherein bacteria
present on the filter are subject to an in situ hybridization when
chemically marking the biological micro particles.
4. The detection method according to claim 1, wherein DNA probes
provided with a fluorescent colorant are used when chemically
marking the biological micro particles, and the fluorescent
colorant is excited by a light source and the light emitted due to
the excited fluorescent colorant is detected by a light detector
when detecting the radiation correlating to the probes.
5. The detection method according to claim 4, wherein the light
intensity is measured by the light detector.
6. The detection method according to claim 5, wherein a photo
multiplier is used for the light detector.
7. The detection method according to claim 4, wherein light
radiation or light pulses and the position of their origin on the
filter are detected using a space-resolving light detector, and the
light pulses and/or the position is counted using a data processing
system.
8. The detection method according to claim 1, wherein the total
concentration of bacteria of a plurality of bacterial species is
detected by the use of correspondingly unspecific DNA probes that
match with nucleic acids or mRNA of different bacterial
species.
9. The detection method according to claim 1, wherein several
different bacterial species are configured to be detected at the
same time by the use of a mixture of differently marked probes.
10. The detection method according to claim 1, wherein the
biological micro particles from the air or from other gaseous media
are collected in a liquid and are passed together with the liquid
over the filter.
11. The detection method according to claim 1, further comprising
performing a PCR process.
12. The detection method according to claim 11, wherein an in situ
PCR process is performed for the subsequent detection of viruses
when performing the PCR process.
13. The detection method according to claim 11, wherein reporter
molecules are released during amplification that are detected due
to their fluorescence by a photo multiplier in accordance with the
PCR process when performing the PCR process.
14. The detection method according to claim 11, wherein an in situ
hybridization is carried out such that after fixing the bacteria
DNA probes are added, and thereafter non-bound DNA probes are
washed away and bound DNA probes are dissolved by mRNA and
amplified by PCR when chemically marking the biological micro
particles.
15. The detection method according to claim 1, wherein the sample
is passed through a micromechanical filtering element and/or
through the nitrocellulose membrane that is used as, instead of, or
in addition to the micromechanical filtering element when passing
the fluid containing the biological micro particles through the
filter.
16. The detection method according to claim 15, wherein positions
of the nitrocellulose membrane that are still unoccupied are filled
up with proteins when chemically marking the biological micro
particles.
17. The detection method according to claim 15, wherein the
biological micro particles adhering in the membrane are marked by
antibodies or by marked probes configured to be detected by
radiation.
18. The detection method according to claim 1, wherein the number
of living cells in a sample is measured.
Description
[0001] This application is a divisional of prior application Ser.
No. 12/598,483, filed on Jan. 28, 2010, which is a U.S. National
stage application that claims priority under 35 U.S.C. .sctn.119(a)
to German Patent Application No. 10 2007 021 387.7, filed in
Germany on May 4, 2007, the entire contents of U.S. application
Ser. No. 12/598,483 and German Patent Application No. 10 2007 021
387.7 are hereby incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates to a detection device for detecting
biological micro particles such as for example living or dead
bacteria, viruses, spores, pollen or biological toxins that can be
marked using probes capable of being detected by radiation, in
particular a detection device for detecting living bacteria,
viruses or biological toxins by means of fluorescent nucleic acid
probes or proteins as probes. The invention also relates to a
detection method for detecting biological micro particles such as
for example living bacteria, viruses, spores, pollen and/or
biological toxins in a fluid.
[0004] 2. Background Information
[0005] Previous techniques for detecting living bacteria require a
time-consuming step of cultivation of the micro organisms. This
conventional technique of detection of living cells or cell number
determination of living cells requires an enormous amount of time
and can be performed only in suitable biological laboratories (S1
to S4 laboratories). An alternative technique for the detection of
living bacteria is the in situ hybridization. The in situ
hybridization technique is a standard technique already frequently
applied in molecular biology. There have been numerous publications
on this technique.
[0006] Concerning bacteria detection, the company of Vermicon AG,
Munich, offers ready-to-use detection kits, i.e. ensembles of
chemicals, for different bacterial strain.
[0007] Previous detection methods including in situ hybridization
are described in more detail in WO 01/68900 A2, WO 02/101089 A2, DE
103 07 732 A1, WO 2005/031004 A2, U.S. Pat. No. 6,844,157B2, US
2005/064444 A1, US/20050202477 A1, US 2005/0202476 A1 as well as US
2005/0136446 A1. For further details it is explicitly referred to
these prior art documents.
[0008] Presently however, the in situ hybridization is possible in
the practice only by using a very expensive and highly sensitive
fluorescence microscope.
SUMMARY OF THE INVENTION
[0009] Accordingly, it is an object of the present invention to
provide a method allowing rapid, reliable and uncomplicated
automatic detection especially of living bacteria, but also of
other micro particles and microorganisms, in particular viruses or
biological toxins, that can be marked using special probes.
[0010] This object is achieved by a detection device comprising the
features of a first aspect of the present invention and by a
detection method comprising the steps of a twenty-fourth aspect of
the present invention.
[0011] Advantageous embodiments of the invention form the subject
of other aspects. A beneficial use forms the subject of another
aspect.
[0012] Advantageous embodiments of the invention enable in
particular an automatic live detection of pathogenic bacteria in
drinking water and air.
[0013] The invention particularly provides a device for rapid and
preferably automatic detection of living bacteria, without the aid
of a fluorescence microscope.
[0014] The measuring results are especially suitable for being used
to obtain a correlation to CFU (colony forming units), with the aim
of providing an alternative to the approved time-consuming
cultivation method (which provides the CFU value).
[0015] According to the invention, a filter is provided suitable
for filtering out the micro particles to be detected. The
substances or chemicals serving in the various marking steps to
mark the micro particles for the purpose of detection are provided
for instance by a supply system and are also passed to and
particularly over the filter. By means of a detection system, the
radiation for detecting the probes can be detected and thus also
the micro particles.
[0016] Especially for the detection of living bacteria a
micromechanical filter is used for example. In such a case, a
supply system delivers especially the chemicals to be used in the
individual steps of an in situ hybridization, for instance from
detection kits available on the market for detecting particular
bacteria.
[0017] The supply system can be preferably automatically controlled
via valves and/or pumps, so that the detection method can be
performed fully automatically.
[0018] Preferably, the filter can be used several times by being
regenerated and/or conditioned using suitable chemicals. This is
enabled by correspondingly controlled pumps and valves.
[0019] In particular, the supply system can be a part of a fluidic
system which does not only supply chemicals that are used for
marking and/or for the later washing and cleaning of the device,
but which is also capable of delivering fluids to be measured to
the filter. The measurement of radiation is adapted to the
respective probes that are used.
[0020] In order for the probes to emit radiation, fluorescent
colorants or for instance also Raman labels, quantum dots or the
like can be used. If probes marked with a fluorescent colorant are
used, as this is known also in the in situ hybridization, a light
source for exciting the fluorescent colorant is preferably
employed, for example a laser.
[0021] As a radiation measuring device for the measurement of
radiation correlated to the probes preferably a light detector is
used which is sensitive to a particular fluorescence radiation.
[0022] The light detector may include for example a photo
multiplier, in order to thus detect the intensity of the
fluorescent light. Alternatively or additionally a space-resolving
light detector, for example a two-dimensional space-resolving light
detector can be used. Such a detector is formed for example by a
two-dimensional CCD array. Such a space-resolving sensor is capable
of determining light pulses and the position of their origin on the
filter. Due to the higher resolution and due to the individual
sensor elements an evaluation unit, which is preferably controlled
by software, can be connected for counting e.g. the light spots and
thus the probes and thus the micro particles.
[0023] For filtering out living bacteria, a micromechanical filter
element can be used for example. Preferably employed are
micromechanical filters having a pore size clearly below the size
of the micro particles to be filtered out. A micromechanical filter
for instance has a pore size of less than 0.8 .mu.m, for example
approx 0.45 .mu.m, so that bacteria having typical dimensions of 1
.mu.m are securely retained.
[0024] Other interesting micro particles, such as for example
viruses or biological toxins, partly have smaller dimensions. For
filtering these micro particles, a micromechanical filter having a
small pore size or a different filter, particularly a
nitrocellulose filter, can be used. For this purpose, a
micromechanical filter or a different porous body as a support are
covered with nitrocellulose. The nitrocellulose membrane can be
provided for instance by a web capable of being moved for the
exchange of the effective nitrocellulose membrane, so that a fresh
web piece can be used as a filter for new measurements.
[0025] For the determination particularly of viruses or biological
toxins, proteins detectible by radiation such as antibodies marked
with a fluorescent colorant or marked nucleic acid probes can be
used.
[0026] For the detection of living bacteria, correspondingly marked
DNA probes are preferably employed which attach to the mRNA that is
only provided by living bacteria having a sufficient lifetime.
[0027] Additionally to this probe marking employed in the in situ
hybridization also a PCR module can be used to alternatively or
additionally perform a PCR detection process.
[0028] In the PCR method, which is also employed in criminology,
pieces of DNA are multiplied and thereafter detected.
[0029] If the concentration of micro particles in liquids is to be
determined, for example the number of living bacteria in drinking
water, this liquid can be directly supplied to the detection device
and passed over the filter. If a concentration of micro particles
in gaseous media is to be determined, for example in the air, these
micro particles can be first collected in a liquid, for example in
an upstream collecting device, e.g. a bio sampler, and this liquid
can subsequently be transferred to the detection system, or the
particles can be directly collected from the air by passing the air
through the filter.
[0030] Advantages of the invention and/or advantages of beneficial
embodiments of the invention are as follows: [0031] A rapid,
uncomplicated and sensitive automatic detection of living bacteria
using the detection device is possible at any location. [0032]
Time-consuming steps of cultivating bacteria in a biological
laboratory can be omitted. [0033] A mobile detection of living
cells is possible. This allows for example rapid testing of
drinking water in a vehicle or aircraft or spacecraft or, in
connection with a robot, for civil and/or military purposes. [0034]
The device and the method enable for example rapid examination of
suspect liquids, in order to prevent biological attacks, and thus
contribute to public safety. [0035] In medicine for example, the
device and the method allow a rapid diagnose of bacterial diseases,
without examination through the microscope.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] Embodiments of the invention will now be described with
reference to the attached drawings, wherein it is shown by:
[0037] FIG. 1 a DNA sequence for explaining the basics of in situ
hybridization;
[0038] FIG. 2 a schematic illustration of the principle of in situ
hybridization;
[0039] FIG. 3 a schematic illustration of a rapid detection device
for detecting living bacteria in drinking water;
[0040] FIG. 4 a schematic illustration of a construction example of
a rapid detection device for detecting living bacteria in fluids
using a fluidic system with pumps and valves as well as a detection
chamber with laser and photo multiplier;
[0041] FIG. 5 a schematic drawing of the detection chamber from
above, said chamber including a micro mechanical filter;
[0042] FIG. 6 a schematic illustration of the detection chamber and
of a detector of a further embodiment of a detection device;
[0043] FIG. 7 a schematic illustration of the detection chamber and
of detectors of a further embodiment of the detection device in
which a combination of in situ hybridization and PCR detection can
be performed;
[0044] FIG. 8 a schematic illustration of the detection chamber
according to a further embodiment; and
[0045] FIG. 9 a schematic illustration for explaining the use of
the detection device according to FIG. 8, for detecting viruses and
biological toxins.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0046] A method and a device for the detection of micro particles
that can be marked by probes capable of being detected by radiation
will be more clearly described in the following with reference to
the drawings. The device and the method are particularly suited for
the detection of living bacteria by means of nucleic acid probes or
of viruses or biological toxins by means of proteins including
antibodies for the probes. Before explaining the device and the
method, the general methods for marking such micro particles by
means of such probes will be more clearly explained.
[0047] Drinking water contains different living and dead bacteria.
A part thereof can be pathogenic. Only the living bacteria are
relevant, because these alone multiply and may constitute a health
risk.
[0048] But many conventional detection methods based upon
antibodies or PCR are incapable of differentiating between living
and dead bacteria. In the following, a device and a method will be
described which in fact allow the detection only of the living and
hence the relevant bacteria.
[0049] Previous detection methods for detecting living bacteria
require a time-consuming step of cultivating micro organisms. For
this purpose, the bacterial samples are streaked out on special
nutrient media and are incubated (breeded) at a particular
temperature for a particular time. Depending on the bacterial
species, this cultivation step may continue for several days. After
the end of the incubation period, the colony forming unit (CFU),
the number of bacteria colonies that have formed, is determined.
This conventional technique of living detection or determination of
the number of living cells is extremely time-consuming and up to
present there is no possibility of a rapid and uncomplicated
detection of living bacteria.
[0050] An alternative technique for the detection of living
bacteria is in situ hybridization. But presently this analyzing
method can be carried out only in a very expensive and highly
sensitive fluorescence microscope. Mobile field tests are
practically impossible and qualified laboratory personnel is
required for operating the microscope.
[0051] For the detection of microorganisms additional detection
methods are frequently applied, for example polymerase chain
reaction (PCR) or enzyme-linked immuno sorbent assay (ELISA). But
these methods exclusively determine the number of the living and
dead cells, hence the total number of cells of a sample. The
determination of the number of living cells is not possible with
these methods.
2. Methods for Bacteria Detection
[0052] A) PCR
[0053] The PCR method (polymerase chain reaction) is a method in
which smallest amounts of a DNA section can be multiplied in a
chain reaction (amplification). Today, the PCR method is frequently
used for various proofs by way of particular DNA sequences, for
example: [0054] in criminology or in paternity proof [0055] in
microbiology, for the detection of living and dead bacteria (total
cell number) [0056] in medical diagnose, for the proof of virus DNA
in the blood, or [0057] in evolution biology, for tracing family
relations and lines of ancestry.
[0058] For PCR detection, two short pieces of DNA (primer) must be
present matching the searched DNA strand. The chain reaction
initiated by them performs up to 30 cycles, the amount of DNA being
doubled at each cycle. Due to the detection via genotype, dead and
living bacteria are detected. A detection exclusively of living
cells is not possible with the PCR method.
B) ELISA
[0059] ELISA (enzyme-linked immuno sorbent assay) is a wide-spread
method of detecting individual proteins (antigen) using the
mechanisms of the immune system: If a substance is recognized by
the immune system as a foreign substance, the immune system
produces antibodies docking to the foreign molecule and thus
marking the molecule. This so-called antibody-antigen interaction
is utilized for the ELISA test.
[0060] If a particular protein has to be detected, the matching
antibodies must be known and must have been prepared in advance
using various gen-technological or cell-biological methods. If the
searched protein is present in a sample, the protein will be bound
by the antibodies applied to a substrate. After the
antigen-antibody interaction a reaction controlled by enzymes is
induced which results in a visible ink precipitate. ELISA tests are
very common today in medical diagnose. But they are also used in
many other fields, if individual proteins have to be detected. In
the case of bacteria detection, the antibody recognizes surface
proteins specific to bacteria. Both the living and dead bacteria
(total cell number) are detected.
C) In Situ Hybridization
[0061] Reference is made in the following to FIG. 1. A DNA sequence
10 may be imagined as a "zipper". The "teeth" of this zipper are
the bases adenine (A), cytosine (C), guanine (G) and thymine (T).
The information contained in the DNA is encoded along the "zipper"
in the order of these four letters. Opposite "teeth" always only
form either AT or GC pairs. The sequence ACGCT for example has the
letter sequence TGCGA as a complementary vis-a-vis.
[0062] During the hybridization, the "zipper" is opened by heating,
so that single strands are exposed. Short pieces of DNA, which are
also single-stranded pieces of DNA and which are used as probes,
are now supposed to find their matching counterpart on this long
single strand. This is the point where the zipper closes again
after cooling.
[0063] This can be made visible by means of markings on the probe
(e.g. by means of a fluorescent colorant 11, see FIG. 2). In this
way it is possible to find out whether or not specific sequences,
representing for example particular marker gens, are present in the
examined DNA (see item A: PCR).
[0064] In the case of in situ hybridization, which is explained in
more detail in the following with reference to FIG. 2, the
fluorescence-marked probes 12 do not associate with the DNA, but
instead with the mRNA 14, the so-called "copy" of the DNA. The mRNA
14 is a kind of copy of the DNA. From the information transcribed
into the mRNA, proteins are formed (protein biosynthesis).
[0065] In dead bacteria this mRNA 14 has an extremely short
lifetime (microseconds) and it is instantly degraded after the
dying of the bacteria.
[0066] With the in situ hybridization method the mRNA of living
cells is preserved (technical expression: "fixed") and then
detected, i.e. exclusively the living bacteria are detected (number
of living cells), since dead bacteria do no longer contain
mRNA.
[0067] Since in this method DNA binds to (m)RNA, one speaks of
hybridization.
[0068] Even a combination of in situ hybridization and PCR is
possible. In this case, the DNA of the bacteria which is also fixed
is used for amplification. This method, which is called in situ
PCR, is employed for instance for the parallel detection of viruses
in the sample to be examined.
[0069] In the meantime, the in situ hybridization method has
developed into a standard method which is already frequently used
in molecular biology. For further details concerning the individual
steps of the in situ hybridization and of the other detection
methods herein presented it is explicitly referred to WO 01/68900,
WO 02/101089 A2, DE 103 07 732 A1, WO 2005/031004, U.S. Pat. No.
6,844,157 A1, US 2005/064444 A, US 2005/0202477 A1, US 2005/202476
A1, US 2005/0136446 A1.
[0070] Ready-to-use detection kits with ensembles of chemicals are
available on the market, e.g. from the company of Vermicon AG,
Emmy-Noether-Strasse 2, D-80992 Munich, Germany. However, the
detection kits which are currently available on the market require
a fluorescence microscope, and the chemicals must be professionally
used by qualified personnel.
Embodiments
[0071] On the other hand, a detection device 20 enabling a fully
automated detection of living bacteria without the use of a
microscope is illustrated in FIG. 3. The detection device 20
comprises a detection chamber 22 in which a filter 24 is arranged
for filtering out the micro particles or microorganisms to be
detected from the fluid to be examined.
[0072] For the detection of living bacteria the filter 24 is
constructed as a micromechanical filter and thus includes a
micromechanical filtering element 26. The micromechanical filter
and its filtering element for example have a structure which is
more clearly described and illustrated in the German patent
application DE 10 2006 026 559.9, which is not a prior publication.
For further details reference is made explicitly to DE 10 2006 026
559, the contents of which is incorporated herein by reference.
[0073] The detection device 20 also includes a fluidic system 28
for passing the fluid, e.g. drinking water, to be examined for the
presence of living bacteria 32, to and through the filter 24.
[0074] The fluidic system 28 comprises one or more pumps 34, 35 and
also several motor valves A, B, C, D. The pumps 34, 35 and the
valves A, B, C, D can be automatically controlled by a control unit
(not further illustrated) connected to it, e.g. a computer
including corresponding software.
[0075] Moreover, a part of the fluidic system 28 serves as a supply
system 36 adapted for supplying chemicals for marking the bacteria
32 or other microorganisms or micro particles to be detected by
means of probes capable of being detected by radiation, for example
fluorescence-marked probes 12, to the filter 24. Another part of
the fluidic system 28 serves as a discharge system 38 for
discharging substances and samples from the detection chamber
22.
[0076] More precisely, the supply system 36 of the fluidic system
28 comprises a motor valve A having an outlet A1 and several inlets
A2 to A9. The following table 1 shows the configuration of the
inlets for a practical example.
TABLE-US-00001 TABLE 1 Configuration of the inlets of the motor
valve A in a practical example. Valve inlet A2 A3 A4 A5 A6 A7 A8 A9
20% H.sub.2O reserve PBS sample of antibody wash 0.5M EtoH (inlet
is drinking buffer NaOH free) water
[0077] Two pumps 34 and 35, which are connected in parallel and
which have a different pumping capacity, connect to the outlet A1.
A first pump 34 works for example in a working range of 6 to 70
ml/min, and a second pump 35 works in a working range of 0.1 to 7
ml/min. Depending on the control and on the switching of the pumps
34, 35, precisely dosed amounts of fluids delivered from the motor
valve A can pumped towards the detection chamber 22.
[0078] Following the pumps 34, 35 is a 3/2 directional valve B
enabling the output of the pumps 34, 35 being selectively directed
to one of the two sides of the filter 24. To this end, the
detection chamber 22 is subdivided into two sub chambers 42 and 44
by a partition 40, and the sub chambers are interconnected trough
the filter 24. While the inlet B1 of the valve B is connected to
the outlet of the pumps 34, 35, an outlet B2 of the valve B is
connected to the first sub chamber 42, and a second outlet B3 of
the valve B is connected to the second sub chamber 44. Depending on
whether the first or second sub chambers 42 or 44 are charged, the
filter 24 can be charged with corresponding liquids, cleaned and/or
conditioned. Corresponding flows can also be achieved via the
discharge system 38 in which a first inlet C1 of a 3/2 directional
valve C is connected to the first sub chamber 42 and a second inlet
C3 is connected to the second sub chamber 44. The 3/2 directional
valve C selectively allows the first inlet C1 or the second inlet
C3 being switched to an outlet C2. The outlet C2 is connected in
turn to an inlet D1 of an additional 3/2 directional valve D. This
inlet D1 can be selectively switched by the valve D to a drain 46
for discharging antibodies or to a manifold 48 for collecting
antibodies.
[0079] That means that by the arrangement of openings for supplying
liquid on both sides of the filter and of openings for discharging
liquid on both sides of the filter and also by the arrangement of
the valves B and C, a flow through the filter 24 in both directions
is achieved. By the switchable valve A, which is connected to two
or more liquid tanks, chemicals for marking, cleaning or
conditioning can flow through the filter.
[0080] For the detection of the bacteria 32 marked with the
hybridization probes or with fluorescence-marked proteins or
antibodies, a radiation measuring device 50 is connected to the
detection chamber 22.
[0081] To detect for instance bacteria 32 which are marked using
fluorescence-marked probes 12, a light source in the form of a
laser 52 is required which emits radiation suitable for exciting
the fluorescent colorant 11, for example light having a wavelength
of 405 nm. This limitation of the wavelength can be further
improved by an optical filter connected downstream of the light
source.
[0082] In the illustrated embodiment the radiation measuring device
50 includes a light detector 54 for detecting the radiation which
is used here, namely fluorescence light 56 from the bacteria 32.
For a more precise detection of such radiation, the radiation
measuring device 50 further includes an optical filtering element
58 which only allows the radiation to be measured, in the present
case the fluorescence light 56 to be measured, e.g. fluorescence
light at a wavelength 455 nm, to pass through.
[0083] For the connection of the radiation measuring device 50, the
detection chamber 22 is closed on one side with a quartz glass 60.
Excitation by means of the laser 52 and light detection by means of
the light detector 54 take place through this quartz glass 60.
[0084] FIG. 4 shows a general drawing of a more special first
embodiment of the detection device 20, wherein the detection
chamber 22 and the fluidic system 28 are only schematically
indicated. In the embodiment illustrated in FIG. 4, the light
detector 54 includes a device for measuring the intensity of light,
in the present case a photo multiplier 62. In the present case a
fluidic system 28 including only a single pump 34 and a simplified
discharge system 38 is used.
[0085] The detection device 20 shown in the FIGS. 3 and 4 allows a
fully automatic detection of living microorganisms using the method
of in situ hybridization. Within this system, living bacteria 32
are rapidly and reliably detected for the first time using the in
situ hybridization method. The core of the device or of the
detection device 20 is the detection chamber 22 containing the
micromechanical filter 26. Through the fluidic system 28 (pumps 34,
35, passages or hoses and valves A, B, C, D) connected to the
detection chamber 22, the sample to be examined (e.g. water 30
containing bacteria 32) is pumped over the filtering element
26.
[0086] Since the micromechanical filtering element 26 has a pore
size of e.g. 0.45 .mu.m and since the bacteria 32 have a diameter
of e.g. 1 .mu.m, the bacteria 32 will accumulate on the filter
surface.
[0087] After their accumulation, the bacteria 32 are prepared for
the actual detection. To this end, the chemicals required for the
in situ hybridization are pumped over the filtering element 26 with
the aid of the fluidic system 28, especially with the aid of the
supply system 36.
[0088] The actual detection is performed using a light source, e.g.
the laser 52, which excites the fluorescent colorant 11 with which
the DNA probe 12 has been marked. The fluorescence light 56 emitted
as a result of the excited fluorescent colorant 11 is subsequently
detected by means of the photo multiplier 62 (PMT). Special optical
filters--in the present case the optical filtering element
58--ensure that the photo multiplier is reached exclusively by
fluorescence light 56 emitted from the marked bacteria 32.
[0089] FIG. 5 is a schematic illustration of the detection chamber
22 including the filter 24 as viewed from above. The grid that can
be recognized represents the micromechanical filtering element 20.
Also shown are connections 64 and 66 to the supply system 36
respectively the discharge system 38 of the fluidic system 28.
[0090] Target organisms for a living detection are for example
pathogenic germs. Due to the base sequence of the
fluorescence-marked probe 12, it is not only possible to quite
specifically detect a pathogenic bacterial species (e.g. E. coli),
but it is also possible to detect bacteria on a higher taxonomic
level. Hence, in the case of E. Coli, it might be possible that all
coli species in a sample are detected.
[0091] By designing the base sequence of the DNA probe 12 to be
more unspecific, it may become possible to detect on an even higher
taxonomic level, e.g. to detect many enterobacteria.
[0092] Even a simultaneous detection of different bacterial species
is possible. To this end, a mixture of differently marked probes 12
can be employed, using a specific fluorescent colorant 11 for the
respective species of bacteria. In this way, it can be determined
which bacteria 32 are present in the concentration, if excitation
takes place at different wavelengths.
[0093] FIG. 6 schematically shows the detection chamber 22
including the filter 24, and the light detector 54 of a further
embodiment of the detection device 20. In this embodiment according
to FIG. 6, the light detector 54 does not include a photo
multiplier, but a space-resolving detector, namely in the present
case a two-dimensional space-resolving detector in the form of a
two-dimensional CCD array that is connected to an evaluation unit,
for example a computer system 70.
[0094] If the detection is not made through a photo multiplier 62,
but two-dimensionally by means of a two-dimensional space-resolving
detector, e.g. a CCD array 68, plane above the filter 24, the
position of the individual bacteria 32 on the filter 24 can be
determined. The presentation or resolution on the CCD array can be
improved by optics comprised of optical lenses.
[0095] By using suitable software, the individual bacteria 32 can
be counted. This allows the actual number of the bacteria 32 being
determined more precisely than by measuring with a photo multiplier
62, because in the latter only the total light intensity is
measured.
[0096] From the light intensity obtained by the photo multiplier 62
the number of bacteria 32 that had been present is derived through
corresponding calibration. Compared thereto, the detection system
illustrated in FIG. 6 is capable of immediately outputting the
number of bacteria.
[0097] The inaccuracy of a measurement through light intensity is
the higher the more different is the concentration of mRNA 14 in
different bacteria 32.
[0098] To detect bacteria in the air or in other gaseous media
using the above-described embodiments of the detection device 20,
these can be collected for example automatically through a suitable
collecting device in a liquid and supplied together with the liquid
to the detection device 20, for example through the connection A6.
Suitable detection devices are available on the market for example
under the name Biosampler from the company of SKC Inc., Eighty
Four, Pa., USA.
[0099] Alternatively, the air may be directly passed through the
filter, in order to accumulate the particles on the filter.
[0100] The detection method that can be carried out with the
detection device 20 is capable of replacing the current certified
cultivation methods for CFU determination. To this end, the probes
12 which are used are so selected that the bacteria 32 detected
with these probes are similarly representative as the bacteria
detected by the conventional CFU determination.
[0101] FIG. 7 still shows in a schematic representation the
detection chamber 22 including the filter 24 and the radiation
measuring device 50 of a further embodiment of the detection device
20.
[0102] The embodiment according to FIG. 7 includes an additional
module 72--here in the second sub chamber 44--for PCR detection of
DNA sequences. The additional module 72 is for example a device for
carrying out a real time PCR. Such devices are available on the
market, for example from the company of Fluidikm Corp., South San
Francisco, USA.
[0103] The additional module 72, which is employed in addition to
the actual detection device 20, enables a combination of in situ
hybridization and PCR. A so-called in situ PCR can be used for
example for a subsequent detection of viruses. The actual detection
could be made using the method of real time PCR. Thereby, reporter
molecules are released during the amplification and due to their
fluorescence the reporter molecules can be detected by a photo
multiplier.
[0104] To this end, the additional module 72 is equipped with a
separate radiation measuring device for measuring fluorescence
light or it utilizes the correspondingly constructed radiation
measuring device 50 of the detection device 20.
[0105] The embodiment illustrated in FIG. 7 particularly enables
the special embodiment of the detection method more clearly
described in the following being carried out. A combination of in
situ hybridization and PCR are performed. In the in situ
hybridization DNA probes 12 are added after fixing the bacteria 32.
The non-bound DNA probes 12 will subsequently be washed away. If
the bound DNA probes 12 are now resolved again by the mRNA 14 and
amplified by PCR, living bacteria detection can thus be made which
even allows detecting extremely small bacteria concentrations.
[0106] Hence, the PCR method here not only serves for determining
the total number (of dead and living bacteria). On the contrary,
the DNA probes previously bound to the mRNA 14, which are only
present in living bacteria having a sufficient life time, and
multiplied many times by the PCR are detected.
[0107] FIG. 8 shows the detection chamber 22 and the filter 24 of a
further embodiment of the detection device 20 which is also capable
of automatically detecting viruses and biological toxins. To this
end, a membrane, particularly a nitrocellulose membrane 74, is
provided. The nitrocellulose layer 74 can be present for example on
any permeable body serving as a support. In the embodiment of the
detection devices 20 illustrated in the FIGS. 8 and 9 the
nitrocellulose membrane 74 is provided as a layer over the
micromechanical filtering element 26.
[0108] FIG. 9 illustrates the principle of the filtration of
viruses 76 and biological toxins 78 by means of the nitrocellulose
as well as the detection by means of fluorescence-marked antibodies
as probes which are used for the detection in this detection
method.
[0109] Accordingly, the FIGS. 8 and 9 more clearly explain a
further application of the rapid detection device 20 for the
detection of pathogenic viruses 76 and biological toxins 78.
[0110] Viruses 76 are small particles (25.about.500 nm) consisting
of a protein shell and--depending on the species of the virus--a
RNA or DNA genome. They do not have a metabolism of their own, but
propagate exclusively through living cells. Known examples are
influenza viruses, ebola viruses or smallpox viruses.
[0111] Biological toxins 78 are extremely stable proteins which are
mostly formed from bacteria, unicellular organisms or plants as
metabolic products.
[0112] These toxins are frequently released into the surrounding
medium where they become enriched in high concentrations. In the
military field biological toxins can be used as biological
weapons.
[0113] Known representatives of biological toxins are
botulinumtoxin (botox) and ricin that can be obtained from the
seeds of the castor plant.
[0114] Due to their small size, viruses 76 and biological toxins 78
can accumulate only to a limited extent on micromechanical filters.
By covering the micromechanical filtering element 26 or a different
permeable body, which serves as a support, with a suitable
absorbing material, especially nitrocellulose 74, viruses 76 and
biological toxins 78 may nevertheless be successfully filtered.
[0115] Nitrocellulose exhibits great affinity towards proteins and
nucleic acids. Due to their surface condition, proteins are
irreversibly bound to the nitrocellulose membrane 74. In this way,
whole viruses with their surface proteins can be fixedly bound to
the nitrocellulose membrane 74.
[0116] Binding proteins and whole viruses to nitrocellulose is a
widely spread method in the field of biological research and is
described in numerous publications ("Westernblot" or
"Dotblot").
[0117] After the viruses 76 have become adhered to the diaphragm
74, the positions of the membrane which are still unoccupied can be
filled up with particular proteins (e.g. BSA bovine serum albumin)
82. This process is called blocking the membrane. After the
blocking the actual detection with specific antibodies 80, which
have been marked with fluorescent colorant 11, takes place.
[0118] Due to this special property of the nitrocellulose it is
possible to suck the sample and all the liquids required for
detection through the membrane 74.
[0119] Should sucking through the membrane 74 be restricted for
some reasons or even be impossible, the membrane 74 could also be
superficially washed using the detection device. This would merely
require a different switching sequence of the 3/2 directional
valves B, C which are positioned next to the detection chamber 22
on the left and right sides thereof (FIG. 4).
[0120] All steps of all of the above-described detection methods
can be carried out fully automatically with the present device--the
detection device 20--that has been developed, so that analyses are
possible also without specially trained personnel.
[0121] Other than for the examination of liquids a device equipped
with nitrocellulose could also be used for the examination of air.
If viruses 76 or toxins 78 present in air shall be detected, the
same automatic program as for liquids can be used, with air or any
other gaseous medium being pumped through the filter.
[0122] For making numerous measurements one after the other, the
membrane 74 can be provided in the form of an endless roll of
nitrocellulose, a unit of which is paid off after each measurement,
thus ensuring that the filter 24 is fresh prior to each
measurement.
* * * * *